Our results suggest the predictive value of systemic inflammation and suggest a potential role of PCT in predicting a poor outcome in mNSCLC receiving PD-1/PD-L1 blocking mAbs. This finding also suggests a potential role of subclinical bacterial infections in defining the response to PD-1/PD-L1 blocking mAbs that deserves further and more specific investigations.Peripheral-immune-checkpoint blockade (P-ICB) with mAbs to PD-1 (nivolumab and pembrolizumab) or PD-L1  alone or combination with chemotherapy represents a novel active treatment for mNSCLC patients. However, this therapy can be associated to immune-related adverse events (irAEs) and high cost. Therefore, finding reliable biomarkers of response and irAEs is strongly encouraged to accurately select patients who may potentially benefit from the immuno-oncological treatment. This is a retrospective multi-institutional analysis performed on ninety-five mNSCLC patients who received real-world salvage therapy with nivolumab or atezolizumab between December 2015 and April 2020. The outcome of these patients in term of PFS and OS was evaluated in comparison with different serum levels of C-reactive protein (CRP), Erythrocyte Sedimention Rate (ESR) and Procalcitonin (PCT) by performing Kaplan–Meier and Log-rank test and multivariate analysis. We found that high baseline levels of CRP, ESR, and PCT were strongly predictive of poor outcome (P <0.05) with the worse prognosis detected in those patients with a baseline levels of both ESR and PCT over the pre-established cut off (median OS recorded in patients with no marker over the cut off  Peripheral immune-check point blockade (P-ICB) with mAbs to the programmed cell death receptor-1 (PD-1) (nivolumab and pembrolizumab) and PD-Ligand (PD-L1)  alone or in combination with chemotherapy is a promising treatment option for metastatic non-small cell lung cancer (mNSCLC) .These innovative immune-oncological strategies are often very effective in the management of mNSCLC patients; however, they may be hampered by frequent more or less severe immuno-related adverse events (irAEs) and rising costs. Additionally, their fast-track evaluation in controlled trials has allowed their introduction in the clinical practice leaving a large amount of questions to be addressed , 4.This treatment has substantial differences by other anticancer strategies like chemotherapy, radiotherapy and molecular target therapy that exert a direct cytotoxic/cytostatic effect on the tumor. On the other hand, PD-1-ICB does not act on the tumor cells but they rather rescue the antitumor activity of tumor infiltrating cytotoxic T cells (CTLs) mostly attenuated as consequence of PD-1 binding to PD-L1/2 in the tumor , 6.In this context, micro-environmental conditions related to chronic inflammation and/or relapsing infections might greatly affect the efficacy of these immune-effectors at several levels. These conditions may induce CTL exhaustion or promote the synthesis of pro-inflammatory cytokines like Interleukin (IL-6) and chemokines that, in turn, can have the following effects: i) to hamper the CTL-mediated response; ii) to enhance the production of immunosuppressive cell lineages  and iii) to promote the activation of multiple peripheral and central immune-checkpoints including those related to the hypoxic induced factor (HIF) and the adenosine receptor pathway –10.This events might be of critical interest in mNSCLC patients who often present coexisting subclinical inflammatory and/or infectious conditions eventually related to the smoking habit, to chronic pulmonary obstructive disease symptoms and to the presence of relapsing infections within the low airways –13.On these bases, we believe that the study of both inflammatory and infection markers in mNSCLC patients addressed to receive immune-checkpoint blocking mAbs could have a key role in understanding their effective interference in CTL activation and consequently on the outcome of these patients. At the present, the majority of the studies in the literature rely on the prognostic role of unspecific inflammatory markers such as white cell counts, NLR, CRP, ESR, LDH or more sophisticated techniques including tumor immune-profiling or microbiology studies –17.Therefore, we have hypothesized that the presence of inflammatory markers mostly associated to bacterial infections of the low airways such as procalcitonin (PCT), might be easily correlated to the clinical outcome of mNSCLC patients subjected to immuno-oncological treatments. PCT, is a 116 amino acid peptide physiologically synthesized by thyroid parafollicular C cells that contribute to maintain the calcium homeostasis once converted to the calcitonin hormone and released in the blood stream –20. In tAt the same time, preliminary reports show that PCT in NSCLC could be correlated to survival, with a worse survival in patients with a PCT >0.1 ng/ml –33.On the light of all these considerations, we carried out a retrospective study aimed to investigate whether the blood levels of PCT compared with conventional inflammatory markers such as CRP and ESR may predict the outcome of mNSCLC patients receiving PD-1/PD-L1 immune-checkpoint inhibitor mAbs.This work is part of a retrospective real-world evidence (RWE) multi-institutional database study including 95 chemo-refractory mNSCLC patients consecutively enrolled to receive salvage therapy with anti-PD-1 (nivolumab) or anti-PD-L1 (atezolizumab) mAb at the OU-RC, and ROU-SI between September 2015 and April 2020 with a median follow-up time of 28 months –36.real world setting as recommended by the international guidelines and regulatory agencies following the standard procedures of administration for each drug. All patients according to their specific disease received: nivolumab (intravenous infusion of 3 mg/kg every two weeks) (84 patients) or atezolizumab  (33 patients) until disease progression or occurrence of severe adverse events. All patients were fit for treatment with no heart, kidney, and liver failure, no alterations in the blood cell counts and no clear sign of infection. All of the patients aimed to receive the treatment presented a good performance status ≤1 according to the Eastern Cooperative Oncology Group (ECOG). A complete physical examination report, histological sampling, hematologic, biochemical, immune-biological, radiological, and instrumental monitoring were available at baseline. Clinical history, physical examination, and record of adverse events were reported prior to each treatment cycle. A CT scan was performed at baseline and repeated every 3 months or in any case of suspected progressive disease (PD). CT scans were evaluated according to the immune Response Evaluation Criteria in Solid Tumors (iRECIST 1.1)  of the World Medical Association and respect of their privacy. All patients received PD-1/PD-L1 blockade in IST 1.1) .All patients were monitored for blood cell counts, biochemistry, CRP, ESR, and PCT before each treatment course and were also monitored for their adrenal hormone profile, ACTH, TSH, thyroid hormones, anti-thyroid auto-antibodies (AAbs), extractable nuclear antigen antibodies (ENA), anti-nucleus antibodies (ANA), anti-smooth cells antibodies (ASMA), and c/p- anti-neutrophil cytoplasmic antibodies (ANCA) each month from the beginning of treatment as reported in previous study –40. OnlyIn order to perform a statistical correlation among continuous parameters and outcomes, we determined different cut-off for survival analysis , on the overall population.The PCT threshold chosen (0.1 ng/ml) was determined on the recent analysis from Kajikawa et al. , as it iTime to events was analyzed with the Kaplan–Meier method and statistics was performed by the log-rank test. Median survival and 95% confidence intervals were reported. Median follow-up was estimated with the reverse method. Hazard ratios (HRs) and their 95% confidence intervals were estimated through the Cox regression proportional model.In the multivariate approach, a forward stepwise procedure was used and the enter and remove limit set to 0.05 and 0.10, respectively. Significant parameters at multivariate analysis were used to build a final model of survival, identifying different subgroups of patients. Chi-Square analysis was used to test the differences among the subgroup identified in terms of clinical variables.Statistics were performed by the SPSS software 23.0 .Our retrospective analysis was performed on a cohort of 95 patients with mNSCLC in our database who presented a parallel monitoring of serum CRP, ESR, and PCT values. In our series there were 77 males and 18 females who had been consecutively enrolled to receive salvage therapy with nivolumab (69 cases) or atezolizumab (26 cases) between November 2015 and April 2020 with a median follow of 28 months.Patients’ demographic and clinical characteristics are summarized in All of the patients were fit for the immunological treatment and presenting an ECOG performance status in a range of 0–1; no clinical or radiological sign of active infection at baseline and no major impairment of heart, liver and kidney functions were recorded.Mean age was 66 years ± 9.9 years, median age 67 years, range 32–85 years.vs. atezolizumab, p: 0.378)  months, with no statistical differences correlated with the treatment (nivolumab : 0.378) Figure 1Haematological parameters mean value and standard deviations were as follow: PCT 0.21 ± 0.50 ng/ml, ESR 44.2 ± 31.4 mm/H, CRP 3.3 ± 5 mg/dl. All the evaluated parameters are reported in vs. >16 mg/L showed a median OS of 19.3 ± 0.6 (mean: 30.5 ± 4.7) vs. 9.7 ± 1.4 (mean: 15.9 ± 2.7) months, respectively, with a p-value of 0.033. Additionally, patients presenting baseline values of ESR≤ vs. >40 mm/h showed a median OS of 19.8 ± 1.5 (mean: 36.5 ± 5.4) vs. 9.8 ± 1.9 (mean: 17.7 ± 3.5) months, respectively, with a p-value of 0.01. Finally, patients presenting baseline values of PCT ≤ 0.1 vs. >0.1 mg/L showed a median OS of 19.6 ± 0.4 (mean: 31.0 ± 4.3) vs. 7.3 ± 0.6 (mean: 10.1 ± 1.4) months, respectively, with a p-value of 0.002.We carried out a statistical analysis aimed to compare the outcome of the patients presenting CRP, ESR and PCT baseline values below or over the respective pre-established cut-off value. The overall survival was obtained for each specific marker  and PCT  were statistically significant. ESR and PCT were used to build a model of OS, dividing the cohort in three subgroups with the relative characteristics summarized in Our retrospective multi-institutional analysis performed in real world setting and aimed to evaluate the effects of either inflammatory and/or infection markers in mNSCLC receiving PD-1/PD-L1 blocking mAbs, showed a direct correlation of CRP, ESR and PCT baseline values with a poor outcome in terms of survival. The PCT threshold chosen was determined on the recent analysis from Kajikawa et al. , as it iThese results, however, are in line with our previous results and with what reported by several authors concerning the negative influence of inflammation on the outcome of patients subjected to palliative chemotherapy, radiotherapy and/or immunomodulating treatments for mNSCLC. On the basis of these data, it can be hypothesized that a coexisting chronic inflammation in mNSCLC patients may affect the immune-balance between the immune-system and cancer growth affecting the anti-tumor activity of T cells and promoting the mechanisms of cancer immune-escape –41.At the same time, additional analysis on the correlation between inflammation parameters and outcomes in the specific setting of immunotherapy is needed, in order to investigate whether these parameters are simply prognostic biomarkers (i.e.: they are correlated to the prognosis of lung cancer patients independently from the choice of the systemic therapies) or predictive biomarkers of response to immunotherapy.regs and MDSCs) and to the triggering of the activation of multiple immune-checkpoints and immune-suppressive adenosine receptors  compared with the outcome recorded in patients with just one of the two marker values over the cut-off (mean OS = 20.1 ± 4.0 months) or in patients with both marker values below the cut-off (mean OS = 40 ± 5.9 months). This finding suggests that both inflammation and infection have an additive and independent detrimental effect on the outcome and on the treatment response to the immuno-oncological treatment with anti PD-1 and PD-L1 blocking mAbs.PCT is considered a serum biomarker able to distinguish bacterial infection from other causes of infection or inflammation. This could be of particular interest in patients with infections of the lower respiratory tract where PCT may help in resolving diagnostic uncertainty and guiding to antibiotic therapy; in details, antibiotics’ discontinuation in patients with pneumonia, is commonly recommended on defined PCT thresholds  in combination with clinical judgment , 52, 53.Streptococcus pneumonia and Haemophilus influentia, are commonly associate to higher PCT levels  compared with atypical bacteria , or other eukaryotic parasites ,  and viruses   , 52. On However, unspecific PCT rises have been sporadically reported upon treatment with immunomodulatory treatments such as T-cell antibodies, alemtuzumab, IL-2, and granulocyte transfusions even though in our setting we were unable to show any significant increase in CRP, ESR and PCT related to the use of mAbs to PD-1 or PD-L1 –56.Serum PCT blood levels commonly rise within few hours after the microbiological insult and roughly correlate with the severity of infection declining at a predictable fast rate with complete resolution. However, it should be taken in consideration that PCT production continues maintaining a plateau level when the infection/inflammatory stimulus is not completely solved –59. On tAs an additional consideration, it is important to take in consideration the in mNSCLC patients receiving immuno-oncological treatments the role of the specific broncho-pulmonary microbiota that in analogy with what reported for the intestinal resident bacteria, may affect the immunological response of the host to the tumor and consequently the efficacy of immune-checkpoint inhibitors. In this context, it has been recently shown that the unspecific use of antibiotics prior to PD-1 blockade is correlated with a poor response to the treatment and it is commonly not advised, although some concerns of bias related to the selection of patients –71. In tAlso, we did not find any correlation between the inflammation biomarkers and the development of irAEs. At this regard, we can speculate that basal parameters cannot predict the immune-related side effects, unfortunately, as immunotherapy has not started yet. We believe that the dynamic evaluation of these biomarkers during the course of immunotherapy  could help in this prediction. A proper investigation of this phenomenon is needed in the next future with a dedicated prospective trial.Our work recognizes the limitation of a monocentric retrospective study that deserves validation in external datasets and/or prospective validation. Presently, external validation was not possible, as PCT is not included in the routinary clinical management of mNSCLC.In the present study we describe the detrimental effect of systemic inflammation and infection in mNSCLC receiving PD-1/PD-L1 blockade immunotherapy and suggest a perspective investigation of CRP, ESR and PCT as potential biomarker of response to the immuno-oncological treatment. The results of this study also open a new research scenario potentially played by bacterial influence and eventually on the preventive use of antibiotics in patients with high baseline levels of ESR and PCT, aimed to receive immune-checkpoint blockade.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.VN, DG, MC, and PC conceived and designed the study and wrote the manuscript. RG, GB, PT, PP, AF, SM, and PC acquired the clinical data. DG performed statistical analysis. VN, LM, LP, AG and PC followed the patients, including planning clinical visits, blood sample collection and follow-up. AL, MC, PC and SZ revised the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ex-vivo skin permeation studies through the rat skin showed that the cumulative amount of imatinib permeated from imatinib-TFS-Gel was significantly higher than that from imatinib-Gel. The RA rat model indicated a substantial reduction in paw edema during the 14 days study following the application of imatinib-TFS-Gel as compared with imatinib-Gel. Therefore, imatinib-TFS-Gel can be considered as a promising drug delivery system for the treatment of RA. In the present study, imatinib-loaded transfersomal gel (imatinib-TFS-Gel) was developed to minimize the oral dosing frequency and side effects during rheumatoid arthritis (RA) therapy. Imatinib-loaded transfersomes (imatinib-TFS) were prepared by the film-hydration method. The effects of lecithin content, lecithin/ EA ratio, and the type of EA on the characteristics of the imatinib-TFS were studied using a D-optimal design. Morphology of imatinib-TFS was investigated using scanning electron microscopy. The optimized imatinib-TFS formulation was used to prepare imatinib-TFS-Gel with the aid of Carbopol 940 as the gelling agent. The Optimized imatinib-TFS had a spherical shape with the particle size of 140.53 ± 0.87 nm, polydispersity index of 0.44 ± 0.01, the zeta potential of -17.63 ± 0.65 mV, encapsulation efficiency of 98.70 ± 0.38%, and release efficiency of 81.26 ± 0.70 %.  It is caused by the invasion of immune cells into the synovium, followed by proliferation of synovial lining cells and development of pannus . As a seMaterialsImatinib base was kindly gifted by Parsian Pharmaceutical Co. (Iran). Soybean lecithin and the dialysis bag (molecular cut off 12000 Da) were purchased from Sigma (USA). Carbopol 940, Span80, Tween 80 and Span 20 were obtained from Merck (Germany). All other used chemicals were of analytical reagent grade.Preparation of imatinib -TFSlecithin (25-50 mg) and Tween80, Span 80 or Span 20 as EA at the ratios of 4:1, 7:1 and 10:1 were dissolved in 5 mL chloroform. Solvent removal under vacuum using a rotary evaporator  produced a thin film. The film was hydrated with 5 mL deionized water while rotating at 180 rpm for 30 min at 60 oC. The dispersions were sonicated at 40 W using a probe sonicator  at pulsed on-time 2 sec, off-time 2 sec for 2 min. Imatinib-TFS were prepared by the thin-film hydration method. In brief, 5 mg of imatinib, different amounts of Experimentaldesign ® Software  was utilized to generate the design and analyze the results, further (24 h%) as described in To assess the effects of lecithin content, lecithin/ EA ratios, and the type of EA on the vesicles properties, a 3-factor, and 3-level D-optimal design were used. Experimental factors and factor levels were chosen based on the results from preliminary studies . Design  further . The stuPhysicochemical characterization of imatinib-TFSPS, PDI and ZP analysisThe PS, PDI and ZP of imatinib-TFS were measured by a Zetasizer . Samples were diluted 5 folds with deionized water before analysis. Determination of the EE% A volume of 0.5 mL from each formulation was centrifuged  at 14000 rpm for 20 min using Amicon® microcentrifugation tubes . The free (un-encapsulated) imatinib was measured in the filtrate by a UV spectrophotometer  at 260 nm. The EE % was calculated from the following equation: Equation 1.Drug release studies24 h% was determined using the following equation:A volume of 0.5 mL from each imatinib-TFS formulation was placed in a dialysis bag, then immersed in 25 mL phosphate buffer solution  containing 0.5% Tween 80. The release medium was kept at 37 °C and stirred at 500 rpm. At predetermined time intervals, 0.6 mL samples from the release medium were withdrawn and immediately replaced with fresh PBS. The amount of released drug was determined by UV spectrophotometry at 260 nm, as described previously. To compare the efficiency of drug release among various formulations, RE Equation 2.Where y.dt is the area below the release curve up to the time, t, and y100.t reflect the rectangle area defined at the same time by 100% release.Scanning electron microscopy (SEM)The morphology of the optimized imatinib-TFS was determined using SEM . For this purpose, a drop of the imatinib-TFS was spread onto a carbon-coated copper grid to leave a thin film. The film was coated with gold under vacuum, then examined under SEM. Fourier-Transform Infrared (FTIR) Spectroscopy−1.The FTIR spectra of imatinib, drug-free TFS, and optimized imatinib–TFS were recorded by FTIR spectroscopy  using potassium bromide (KBr) pellets technique at room temperature. To form pellets, imatinib, lyophilized drug-free TFS, or optimized imatinib–TFS were thoroughly mixed with KBr. Each KBr pellet was then scanned at the frequency range of 4000–400 cmFreeze-drying of imatinib–TFSoC) for 24 h. The frozen samples were immediately subjected to freeze-drying  at 0.07 mbar for 48 h. Each lyophilized sample was reconstituted in 2 mL purified water by gently shaking. The reconstitution time, PS, and PDI of samples were measured.The impact of 1% and 3% concentrations of sucrose, mannitol, or lactose as cryoprotective agents on the imatinib-TFS stability throughout freeze-drying was examined. The cryoprotectant was added to the formulation. The samples containing dissolved cryoprotectant were placed in a freezer  containing 0.5% Tween 80 and stirred at 500 rpm during the study. The temperature of diffusion cells was kept at 37 ± 0.5 o C using a re-circulating water bath. At predetermined time intervals, 1ml samples were withdrawn from the receiver compartment and replaced with fresh medium. The amount of imatinib was analyzed by UV spectrophotometry at 260 nm. Each test was performed three times. To evaluate permeability, the mean cumulative amount of imatinib permeated per unit surface area of the rat skin was plotted against time and the permeation flux The rat skin was shaved, excised, and rinsed with physiological saline. Skin samples were clamped between the receptor and donor of Franz diffusion cell with a diffusion area of 4.5 cmIn-vivo anti-RA activity of imatinib-TFS-GelAnimalsThe 8–10 weeks old male Wistar rats weighing between 180 and 220 g were used in this study. Rats were housed in cages  and maintained under standard temperature and light/dark cycle. They were fed with a standard pellet diet and had free access to water. The animal experimental study was approved by the Ethics Committee of Isfahan University of Medical Sciences, Isfahan, Iran.RA Induction and monitoringet al. . Group I received imatinib-Gel (0.1% w/v), group II received imatinib-TFS-Gel (0.1% w/v), Group III and IV received imatinib-free gel and imatinib-free TFS-Gel, respectively. Two hundred milligrams of each formulation were administered topically on the paws of arthritic rats once daily. Just before CFA injection and on days 5, 8, 11 and 14 post-injection, ankle diameters were measured using a digital caliper. The percent increase in ankle diameters at each time used was calculated from the following equation: Experimental RA was induced as described previously by Nasr et al. . BrieflyEquation 3.o indicates ankle diameters at day 0 and Dt is ankle diameters at any specified time.Where DOn day 14, animals were sacrificed and the left and right hind paws were transected for determination of their weights. The weight differences between the right (RA) and left  paws were used as another index of CFA-induced paw inflammation. Experimentation designIn this study, imatinib-TFS were prepared using the film hydration method. The D-optimal design was used to optimize and assess the main effects of formulation variables including lecithin content, lecithin/EA ratio and the type of EA on the PS, PDI, ZP, EE% and RE%, as described in Effect of independent variables on PS and PDIp-value < 0.05, et al. . As shown in et al. . The PDI was reversely dependent on the lecithin content and lecithin/ EA ratios . ZP refers to total electrical charges that develop at the interface between the dispersed particles and the liquid medium. The magnitude of ZP determines the degree of repulsion between particles of the same charge, and thus the physical stability of a formulation. As indicated in TFS consisting of Tween 80 showed the highest absolute value of ZP . This cop-value <0.05, The increase in lecithin amount led to the decrease in ZP . As shown in i.e. where the lowest lecithin/ EA ratio is used, may decrease the packing density of the bilayer resulting in the lower EE capability of vesicles. However, further increase of lecithin/ EA ratio from 7 to 10, led to the decrease in the EE. The lower EE at a higher ratio of lecithin/ EA ratio may be related to the size of TFS. Since the lipophilic drug are incorporated within the lipid bilayer, the small size of TFS limits space within the bilayer for large amounts of drug to accommodate , 2931.27 cm-1 , 2789.53 cm-1 , 1644.02 cm-1 (C=O carbonyl), 1583.27 cm-1 (C=C aromatic) and 1552.42 cm-1(C=N aromatic). In addition to characteristic bands which were identified in the FTIR spectra of blank TFS, imatinib-TFS showed absorption bands related to imatinib with no significant shift. The results indicated the entrapment of imatinib into the TFS, as well as no physicochemical interaction between imatinib and excipients in TFS formulation.The FTIR spectra of imatinib, blank TFS, and imatinib-loaded TFS are shown in Freeze-drying of imatinib-TFSThe major obstacle limiting the application of nanoparticles is related to their physical and/or chemical instability; this could commonly occur when there is the storage of these nanoparticle aqueous suspensions for a relatively long period. To improve the physical as well as chemical stability of such systems, the removal of water should be done. Freeze-drying is known as the most widely applied process allowing the conversion of solutions or suspensions into solids with adequate stability for distribution and storage in the pharmaceutical field. Freeze-drying is regarded as an industrial process consisting of removing water from a frozen sample through sublimation and desorption in vacuum conditions. In such a process, different stresses may be generated during the freezing and drying stages. Therefore, protectants are usually added to the formulations to prevent or minimize destabilization processes during freezing and desiccation stresses . The meaet al. . The enhanced drug permeation from imatinib-TFS-Gel could be attributed to nanosized TFS. In addition, the presence of the surfactant in the TFS structure may enhance the penetration of vesicles via rendering deformability to TFS (The y to TFS . TFS cany to TFS In-vitro release of imatinib-TFS-Gelvs. ~100%). The imatinib slower release from TFS-Gel may be attributed to the obstructive impact of the gel matrix (The release profiles of imatinib from optimized imatinib-TFS and imatinib-TFS-Gel over time are shown in l matrix -31. Undel matrix , who shoIn-vivostudiesth days of the treatment (p < 0.05, The CFA subcutaneous injection produced local edema within a few hours . The chath day of the treatment. The increased percentage in paw weight in the RA animals which were treated with drug-free gel, drug-free TFS-Gel and imatinib-Gel was found to be 85.2%, 75.1% and 90.4%, respectively. The least weight increase was 42.9% which belonged to the animals treated with imatinib-TFS-Gel. This was also in agreement with the results related to ankle diameter, both demonstrating the efficacy of imatinib-TFS-Gel in the treatment of arthritic rats. The more therapeutic efficacy in the developed formula could be attributed to the ability of TFS to increase the imatinib percutaneous permeation. So, a higher amount of the drug could be transferred to deeper skin layers, thus improving the therapeutic anti-inflammatory response. These results were also in agreement with the previous research, where, Lei W et al. (W et al.  reported24 h% through D-optimal design. The optimal TFS formulation composed of 27 mg lecithin, lecithin/ EA ratio of 10 and span 80 as EA yielded vesicles of least PS, PDI and ZP and highest EE and RE values. The optimized imatinib-TFS formulation incorporated into the Carbopol 940 gel showed enhanced flux of imatinib through the rat skin as compared to imatinib-gel. Animal studies also exhibited improved paw inflammation in the group treated with imatinib-TFS-Gel, as compared to the group treated with imatinib-Gel. Our results demonstrated that transfersomal gel systems provided enhanced drug permeability through rat skin and improved therapeutic response in RA animals, as compared to conventional drug-incorporated gel systems. However, further studies are required to completely evaluate the promising potentials of such delivery systems for topical delivery of drugs, including imatinib in the treatment of inflammatory skin diseases.In the present study, imatinib-TFS was successfully prepared by thin-film hydration method and optimized for size, PDI, ZP, EE% and REThe authors report no conflicts of interest


Chlamydomonas reinhardtii GY-D55 cells. The principal component analysis (PCA) of transcriptome profiles showed that the samples of the early and late stages were well separated. A total of 12,445 genes were detected as differentially expressed genes. There were 1,861/2,270 common upregulated/downregulated genes for each time point compared with control samples. Samples treated with salt for 2, 8, and 24 h had a relatively large number of characteristic upregulated/downregulated genes. The functional enrichment analysis highlighted the timing of candidate regulatory mechanisms for salt stress responses in GY-D55 cells. Short time exposure to salt stress impaired oxidation-reduction, protein synthesis and modification, and photosynthesis. The algal cells promoted transcriptional regulation and protein folding to deal with protein synthesis/modification impairments and rapidly accumulated glycerol in the early stage (2–4 h) to cope with osmotic stress. At 12 and 24 h, GY-D55 cells showed increased expressions of signaling and photosynthetic genes to deal with the damage of photosynthesis. The co-expression module blue was predicted to regulate endoplasmic reticulum (ER) stress at early time points. In addition, we identified a total of 113 transcription factors (TFs) and predicted the potential roles of Alfin, C2C2, and the MYB family TFs in algal salt stress response.It is highly necessary to understand the molecular mechanism underlying the salt stress response in green algae, which may contribute to finding the evolutionary cues of abiotic stress response in plants. Here, we reported a comprehensive temporal investigation of transcriptomes using data at eight different time points, from an early stage (2 h) to a late stage (up to 96 h) in  Chlamydomonas, can quickly adapt to either climate change-induced or physical abiotic stress. Therefore, it is necessary to explore the molecular mechanism underlying the salt stress response in green algae, which may contribute to identifying the evolutionary cues of abiotic stress response in plants and developing salt-resistant crops with valuable salt-responding characteristics , including the biochemical limits to macromolecular stability and the genetic instructions for constructing macromolecules that stabilize under one or more extreme conditions  TFs play important roles in mediating photosynthesis and lipid accumulation of alga in response to stresses. Several members from bZIP, C3H, Dof, and MYB TFs families were differentially expressed throughout the entire time course in cold stress responding , whereas the growth and photosynthetic activity in several strains of C. reinhardtii recover over 24 h  as progenitor cells   .C. reinhardtii strain GY-D55 (but are expressed only in a short period of time) have not been identified yet, due to the fact that the previous proteomic and transcriptomic studies did not have sufficient temporal resolution. Here we report a comprehensive temporal investigation of transcriptomes using data for eight time points, from early stage (2 h) to late stage (up to 96 h). This time-course transcriptome analysis aims to highlight the timings of candidate regulatory genes and mechanisms for salt stress responses in GY-D55 cells.It is highly possible that many genes that are important for the salt stress responding in the C. reinhardtii strain GY-D55 was obtained from LeadingTec Co., Ltd.1 and maintained in 150 ml of BG-11 medium on a shaking table at 120 rpm under the following conditions: photoperiod 16-8 h/light-dark, temperature 23°C, and light intensity 100 μmol m–2s–1. Under the above-mentioned conditions, C. reinhardtii cells were cultured in a BG11 medium for approximately 14 days until the cell density reached 2 × 106 cells/ml. The cells of the mid-logarithmic phase were used for salt stress treatment referring to the study published by The wild-type C. reinhardtii cells by salt stress at different time points, which helped us design time points. In the study published by C. reinhardtii is initially (1–2 h) blocked by the addition of 200 mM salt. During 4–12 h, the decreasing trend of photosynthetic activities slowed down and respiratory activities began to recover gradually  for 48 h. Besides, 200 mM NaCl treatment inhibits the growth and significantly increases the lipid content of C. reinhardtii at 3 days (72 h)  . Salt trs (72 h) . BesidesC. reinhardtii were collected at eight preset time points after exposure to 200 mM NaCl: 2, 4, 8, 12, 24, 48, 72, and 96 h. To evaluate the morphological changes, including the difference in cell size, salt stress-induced palmelloids, or chlorophyll bleaching under stressful conditions, a 50 μL sample was observed using an Olympus SZ61 microscope  at 40/100× magnification. The t-test analysis was used to evaluate the growth ratio between cells under control and saline condition. The cell count of algal material was estimated at eight time-points and relative growth rates (RGR) were calculated: RGR = (lnN2 − lnN1)/Δt × 100%, where N1 is the cell count at time-point 1 and N2 is the cell count at time-point 2, Δt is the time interval (hours). The statistical differences between salt stress and control samples were analyzed by the one-way ANOVA in R.The NaCl treatment was carried out as previously described by g for 5 min, and the cell pellet was resuspended in 25 ml RNAlater solution (Ambion). The total RNA was isolated using the TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. The integrity of RNA was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system  and the NanoDrop 2000 spectrophotometer .Briefly, 100 ml cell cultures from different groups were subjected to centrifugation at 3,000 ® UltraTM RNA Library Prep Kit for Illumina®  according to the manufacturer’s instructions, and the sequences were attributed to each sample based on the index codes. Briefly, total RNA was subjected to purification using poly-T oligo attached magnetic beads. Purified messenger RNA (mRNA) was reversely transcribed into first-strand complementary DNA (cDNA) using a random hexamer primer and M MuLV Reverse Transcriptase (RNase H–). DNA Polymerase I and RNase H were adopted for the synthesis of the second-strand cDNA. cDNA fragments of 370–420 bp were subjected to purification using the AMPure XP system . The size selected, adaptor-ligated fragments were purified isolated and enriched by PCR. The resulting products were subjected to high throughput sequencing using an Illumina HiSeq X platform . All genetic data have been submitted to the NCBI Sequence Read Archive (SRA) database,2PRJNA770825 and the Gene Expression Omnibus (GEO) database,3GSE191218.Sequencing libraries were constructed using the NEBNext4 Raw reads of the fastq format were firstly processed through in-house perl scripts. In this step, the adapter containing reads, ploy-N containing reads, and low-quality reads were removed from raw data, yielding clean reads. Meanwhile, the Q20, Q30, GC content, and sequence duplication levels of the clean data were determined. All the downstream analyses were conducted using high-quality clean data. Reference genome5 and gene model annotation files6 were downloaded from the genome website directly. The index of the reference genome was built using Hisat2 (v2.0.5), which was adopted to align paired-end clean reads to the reference genome. For the prediction of novel transcripts, StringTie (v1.3.3b) was adopted to assemble the mapped reads of each sample as previously described  was carried out at Novogene Co., Ltd.escribed .C. reinhardtii using a PE150 sequencing analysis. The number of reads mapped to each gene was determined using the featureCounts v1.5.0 p3. Subsequently, the length of the gene and read count mapped to this gene were used to calculate the fragment per kilobase of exon model per million mapped reads (FPKM) of each gene. Differential expression analysis of salt-treated and control samples at different time points was performed using the DESeq2 R package  were defined using thresholds of P-value < 0.05 and | log2(foldchange)| > 0 and 1. The union of the DEGs was analyzed by the heatmap package of R for gene cluster construction, using the Euclidean distance clustering algorithm. The normalized expression values of DEGs were calculated by dividing their expression level at different time points with their maximum observed FPKM.In the present study, 27 independent cDNA libraries were constructed for individual samples of  package . The falP-value < 0.05) were identified.The FPKM values of genes were transformed and normalized to construct the weighted gene co-expression network analysis (WGCNA) co-expression network. The genes with a low variance of expression values among samples were omitted. A co-expression network for the filtered genes was constructed using the WGCNA package in R (Version 3.3.2) . After sArabidopsis thaliana with an E-value cut-off of 1e–5, the genes of C. reinhardtii were mapped to A. thaliana gene IDs accordingly. The TF data of A. thaliana were downloaded from plantTFDB.7 The homologous of A. thaliana TFs in C. reinhardtii were screened as TFs in C. reinhardtii strain GY-D55. The Gene Ontology (GO) enrichment analysis for DEGs of different clusters and genes of co-expression modules was conducted by the topGO package of R.Based on the sequence similarity against the genome of –ΔΔCt method. The statistical differences between samples were analyzed by one-way ANOVA in R. The correlation coefficient of gene expression between qRT-PCR analysis and RNA-Seq data was analyzed in R.Eight different genes were selected to verify the RNA-Seq result, including four upregulated genes  and four downregulated genes . Gene sequences were extracted, and their primers were designed using Primer 5 software . RNA extC. reinhardtii strain GY-D55 was detrimentally affected in the presence of 200 mM NaCl (P < 0.01). In the NaCl-treated group, the RGR was negative during this period, indicating that the growth of GY-D55 cells was significantly inhibited in the early stage of salt stress. During 8–12 h, although the RGR of algae under salt stress was still significantly lower (P < 0.05) compared with the control group, GY-D55 began to gradually increase the number of cells during this period. After 48 h, the RGR of GY-D55 cells under salt stress was higher compared with the control group, although both values were very small. Besides, 200 mM NaCl exposure GY-D55 cells showed the time-dependent formation of palmelloids  of C. reinhardtii strain GY-D55 could be mapped on the C. reinhardtii genome  after alga cells were treated by 200 mM NaCl. Principal component analysis (PCA) indicated an apparent difference in gene expression between samples under control and different time-point salt stress. Clustering analysis using whole gene expression profiles showed an apparent time-dependent similarity in the profiles of the control and NaCl treated samples (R2 = 0.733–0.832) and samples of late time points were highly correlated with each other (R2 = 0.945–0.963).To elucidate the time-course of the transition in the gene expression of the  samples : (1) the samples  that genC. reinhardtii strain GY-D55 under control or salt stress treatments at eight time points were evaluated based on the FPKM values. The relative expressions of genes in C. reinhardtii strain GY-D55, we clustered the 12,445 DEGs into eight clusters using the Euclidean distance clustering algorithm  and low mean connectivity (near 0) . The merThe salt stress treated time points were collected as salt stress responding trait, and module–trait relationships were calculated according to the positive correlation between 44 modules and traits. The module trait relationship is shown in The results of the GO analysis of the filtered salt stress responding related modules were shown in A total of 1,192 DEGs cluster C5 genes were included in WGCNA co-expression module blue, including 17 TFs. Top 1% co-expression links of module blue were extracted for reducing complexity. The five C5 TFs included in the top 1% co-expression links in module blue are Alfin-CHLREDRAFT_127913, C2C2-CHLREDRAFT_205871, C2C2-CHLREDRAFT_171659, MYB-CHLREDRAFT_168936, and RWP-CHLREDRAFT_195915. Alfin-CHLREDRAFT_127913, C2C2-CHLREDRAFT_205871, and MYB-CHLREDRAFT_168936 were predicted to interact with 173, 72, and 6 genes, respectively, some of their potential target genes are overlapped.P-_value < 3.464e–07; the result is shown in To verify the RNA-seq results, an alternative strategy was selected for the upregulated genes. In total, eight genes were selected for validation by qRT-PCR using the same RNA samples that were used for RNA-sequencing . The priC. reinhardtii shows that at the morphological level, salt stress of 150 or 200 mM NaCl results in flagellar resorption, decreased cell size, impaired growth, and palmelloid morphology  at 2–48 h, UGT-80 (CHLREDRAFT_154976) at 2–12 h, and UGP2 (CHLREDRAFT_157049) at 2–4 h in GY-D55 cells suggested the function of these genes in cell wall carbohydrates of palmelloid formation during the early stage of salt stress in microalgae.Existing research on rphology . In the  mM NaCl , especiathaliana . UDP-gluthaliana . BesidesC. reinhardtii recover over 24 h. In the present study, clusters C5, C4, C2, and C3 contained genes mainly expressed at 2–4, 8, 12, and 24 h, respectively  was upregulated at 24 h in the analysis performed by P5CS2 regulated proline synthesis in modulating the salt stress tolerance in GY-D55 cells. In both transcriptomic analyses, the coding gene of glutathione S transferase (CHLREDRAFT_195543) was upregulated under the saline stress in GY-D55 cells, while many genes of the ROS-scavenging system were downregulated. nivalis , C. rein mM NaCl . We comp mM NaCl . In mostI damage  and repa. salina . These r. salina  biosynthC. reinhardtii is initially suppressed in the presence of 200 mM salt, while it recovers over 24 h (a/b protein of photosystem I (CHLREDRAFT_136294). Although the expressions of several PSI- and PSII-related genes slightly recovered after 24 h in GY-D55 cells (SQDG (CHLRE-DRAFT_27658) was reduced in GY-D55 cells at 2–8 h in our study, and it is downregulated at 24 h in the study of SQDG provides a potential mechanism for a key difference between alga’s fatty acid responses, this strain did not display the lipid accumulation response to salt stress with the downregulation of fatty acid desaturase. This finding helped elucidate that what caused salt stress was a lipid trigger, but not C. reinhardtii strain GY-D55.Photosynthetic activity in ver 24 h . The CC-55 cells . PSI has55 cells . Salt ex55 cells . UDP-sulC. reinhardtii . In A. thaliana, MYB88 participates in the stomatal development in abiotic stress responses and the regulation of cell cycle genes . As a member of the HSP70 family, BiP resides within the lumen of the ER, and it helps the folding and assembly of newly synthesized proteins as they are translocated into the ER and can also bind to misfolded, underglycosylated, or unassembled proteins  to cope with osmotic stress. At 12 and 24 h, GY-D55 cells showed increased expressions of signaling and photosynthetic genes to deal with the damage of photosynthesis. The co-expression module blue was predicted to regulate ER stress at early time points. In addition, we identified a total of 113 TFs and predicted the potential roles of Alfin, C2C2, and MYB family TFs in algal salt stress responses.Here, we reported a comprehensive temporal investigation of transcriptomes using data at eight different time points, from early stage (2 h) to late stage (up to 96 h) in PRJNA770825 and the Gene Expression Omnibus (GEO) database, GSE191218.The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number L-YZ and S-JF designed the experiments. L-YZ, Z-TX, and L-QC carried out the experiment and analyzed the data. L-YZ and Z-TX wrote the first draft of the manuscript. L-YZ, X-JZ, and S-JF supervised and completed the writing. All authors revised and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


All prepared membranes were tested for their desalination performance. The membranes containing UiO-66 were found to outperform the blank and the reference counterparts. The reason for this outperformance is possibly attributed to the hydrophilicity of UiO-66 nanoparticles and the presence of nanochannels in their structure. UiO-66 nanoparticles are considered highly potential fillers for the application in desalination membranes. In this study, UiO-66 nanoparticles were anchored to PES membrane substrates, which were subsequently subjected to the interfacial polymerization reaction to coat a layer of polyamide (PA) on their surface. For comparison, a blank membrane incorporating no UiO-66 and a reference membrane incorporating ZrO Materials possessing uniform nanoscale channels are considered next-generation candidates for the application in filtration membranes, for example, carbon-based materials , protein-based counterparts (Aquaporin), and metal-organic frameworks (UiO-66 nanoparticles) . In seve2+ and 99.3% for Al3+) from the water via size exclusion mechanism [Concerning the metal-organic framework (MOF), different candidates have been successfully employed until recently ,7,8. In echanism . 2 (instead of UiO-66) were prepared following analogous procedures. Finally, all prepared membranes were tested for their desalination performance, and their performance results were compared. To the best of our knowledge, compared to the previously reported works given in The superior performance of the MOF materials in filtration membranes had motivated us to carry out different studies in the past ,14. In a4) (purity > 99.9%) and terephthalic acid (purity > 99%) were purchased from Sigma-Alrich. N,N-dimethylformamide (DMF) was obtained from Wako Chemical Industries Ltd. . Polyethersulfone membrane  was received from Millipore. 1,3-Diphenylene diamine (MPD) (purity > 98%), trimesoyl chloride (TMC) (purity > 98%), trimethylamine (TED) (purity > 99%), and (+)-10-camphosulphonic acid (CSA) (purity > 98%) were bought from Tokyo Chemical Industry Co. Ltd. . Hexane (purity > 96%) was delivered from Kanto Chemical Co. Inc.  These chemicals were used without further purification.Zirconium tetrachloride (ZrCl4 (0.30 g) was dissolved in 90 mL of DMF. At the same time, another solution was prepared, which contained 0.22 g of terephthalic acid dissolved in 90 mL of DMF. Thereafter, both solutions were mixed and stirred. To the mixture, 0.18 mL of water were added. The water acted as a modulator that raises the nucleation rate and controls the distribution size of UiO-66 nanoparticles. Subsequently, the mixture was stirred at 100 °C for 20 h, giving a homogeneous and stable dispersion of UiO-66 nanoparticles in DMF. UiO-66 nanoparticles were immersed in methanol for three days. During this time, the solvent was removed, and an identical amount of pure methanol was compensated every day. Prior to the preparation of UiO-66/PES membranes, the UiO-66-containing methanol solution was sonicated for 2 h followed by centrifugation at 2000 rpm for 10 min. Finally, the supernatant was used for the fabrication step of the UiO-66/PES membrane.The preparation of UiO-66 nanoparticles was carried out under a nitrogen atmosphere following our previously documented procedure . BrieflyUiO-66/PES membranes were prepared by a suction filtration set-up following the previously reported procedure . Briefly−1 using a diamond crystal. Transmission electron microscopy  at an acceleration voltage of 100 kV was used to determine the size and morphology of UiO-66 nanoparticles. The UiO-66 dispersion was diluted 100 times in methanol and casted onto a TEM grid. The crystalline structure of the dried UiO-66 nanoparticles was analyzed by X-ray diffraction  using Cu Kα radiation (λ = 1.54 Å) at 40 kV and 30 mA in the interval 5–35°.The functional groups of the membrane surfaces were analyzed by attenuated total reflectance infrared spectroscopy  in the range 450–1800 cmThe morphology of the membranes was observed by scanning electron microscopy  at an accelerated voltage of 20 kV. The hydrophilicity of the membrane was evaluated by measurement of the contact angles on a contact angle meter .The desalination tests were conducted using six identical handmade dead-end stirred cells , which w2. Each filtration cell was equipped with a stirred bar to avoid concentration polarization during filtration. The filtration system was comprised of 6 identical filtration cells . A filtef and Cp represent the concentration of NaCl in the feed and permeate solution, respectively.The permeability (J) and the selectivity (R) were determined via the following equation:−1 were ascribed to the out-of-phase stretching modes of the carboxylate group. On the other hand, the peaks at 475, 548, and 744 cm−1 are attributed to the bending of OH and CH mixed with Zr-O modes as well as Zr-(OC) asymmetric stretching vibration, respectively. The absence of bands assigned to the possible remaining solvents indicates a thorough washing process. Overall, these peaks are in accordance with previously reported results [The ATR-IR spectrum of UiO-66 nanoparticles is depicted in  results .The X-ray diffractogram of UiO-66 is illustrated in 6O4(OH)4 clusters.The TEM image of the UiO-66 nanoparticles is given in The prepared membranes were characterized by SEM analysis . The PESThe wettability of the prepared membranes was evaluated by measurement of the water contact angle (WCA). WCA of the neat PA/PES membrane M1 was 64°. In comparison, WCA of the UiO-66-coated membranes M7-8 was 55.5 and 54.4°, respectively. It indicates that the incorporation of the hydrophilic UiO-66 nanoparticles improved the hydrophilicity of the PA/PES membrane. Additionally, the higher the concentration of UiO-66 nanoparticles inside the membrane matrix, the higher the hydrophilicity of the PA/PES membrane. Generally, the higher hydrophilicity of the membrane is expected to demonstrate higher permeability . 2∙h∙bar and a selectivity of 87.5%  but a higher selectivity. Hence, the presence of UiO-66 nanoparticles improved the selectivity. This improvement was referred in previous studies to the facilitation of mass transport through the nano-sized channels of the UiO-66 nanoparticles [First, the blank membrane M1 was tested for desalination. M1 has a WCA value of 64°. It displays a permeability of 0.64 L∙mof 87.5% b. In comof 87.5%  has highof 87.5%  and surfarticles ,32,33. 2∙h∙bar. Noticeably, the selectivity also increased to 93.6%  compared to membrane M3 (0.42 L∙m2∙h∙bar). The permeability of membrane M4 is even higher than that of the blank membrane (0.64 L∙m2∙h∙bar)  a. It is 2∙h∙bar), it lost more than 25% of the selectivity due to the insufficient presence of UiO-66 nanoparticles and the integrity of the selective layer. Therefore, we increased the integrity of the barrier layer by raising the contact time with the organic phase to 10 s  but remarkably higher selectivity (94.3%) (2∙h∙bar) but lower selectivity (90.5%)  b. FurtheEntry 8, . The exp2 nanoparticles  but a lower permeability (0.88 L∙m2∙h∙bar)  c. This i2/PES membrane. The improvement is possibly due to the hydrophilicity of UiO-66 nanoparticles and the stronger preference of water diffusion through the intra-particle channels. Additionally, the preference is possibly related to the compactness and thickness of the polyamide polymer, which depends on the amount of the monomer and the cross-linker adsorbed on the surface of the substrate membrane. Further investigations into this approach are currently being undertaken for other amine monomers and cross-linkers. To summarize, different PA/UiO-66/PES membranes were prepared by a three-step process. The membranes were characterized by the SEM technique, displaying a homogenous distribution of the particles on the membrane surface. The membranes were also tested for their desalination performance, giving promising filtration results. The membranes incorporating UiO-66 nanoparticles outperformed the blank PA/PES membrane and the reference PA/ZrO


The rainy season in the Philippines is from June to October; this is when the number of dengue cases typically increases. In 2020 during this time, the world was facing the threat of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Coronavirus disease 2019 (COVID-19) and dengue viral infections have similar presentations and laboratory findings, including fever and thrombocytopenia, and there have been reports of coinfection with SARS-CoV-2 and arthropod-borne virus. Here, we report a case of SARS-CoV-2–dengue virus coinfection in the Philippines in a female aged 62 years, whose early symptom was fever and who was positive for SARS-CoV-2 and positive for dengue. Early recognition of such coinfection is important so that proper measures can be taken in the management of the patient. Dengue fever is a mosquito-borne viral infection found mostly in tropical climates, including the Philippines. The clinical manifestations of dengue may include high-grade fever, headache, retro-orbital pain, muscle and joint pains, and rashes. In 2019, the Philippines had one of the highest numbers of reported dengue cases among countries in Asia and South-East Asia. , with associated headache  and retro-orbital pain, generalized body ache, myalgia and arthralgia. There was no associated nausea, vomiting or blurring of vision. The patient had pain over the ankle joints, with no associated warmth or limitation of movement, and no rashes, cough or dyspnoea. The patient had self-medicated with paracetamol, which afforded temporary relief; however, her condition was persistent, with body malaise and weakness, prompting consultation at Baguio General Hospital and Medical Center Emergency Department.The patient was admitted to the COVID-19 ward under the Internal Medicine Service, as a suspected case of COVID-19. The patient denied any history of travel outside the town or direct contact with anyone positive for COVID-19. She reported attending the public market three days before onset of her symptoms. At the emergency department, the initial physical examination of the patient was unremarkable except for decreased breath sound on the right basal lung field. Given the history of fever, the patient was managed as a suspected case of COVID-19.Laboratory tests included reverse transcriptase polymerase chain reaction (RT–PCR) for COVID-19 (Sansure Biotech®), chest X-ray, complete blood count, blood culture, and inflammatory markers such as lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), ferritin, C-reactive protein (CRP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The complete blood count initially revealed leukopenia at 3.16 × 109/L , with haemoglobin and platelet counts being normal . Chest X-ray revealed pneumonia on the right lower lobe of the lung . There was no growth on blood culture. Markers of inflammation were elevated, including ferritin at 2156 ng/mL, ESR at 35 mm/hour, CRP at 18.73 mg/L and LDH at 317.92 U/L. Liver enzymes were elevated, with AST at 100.24 U/L ( × 2.86) and ALT at 65.11 U/L ( × 1.86).In the COVID-19 ward, the patient’s social and environmental history was further investigated. A suspicion of dengue fever was considered after a comprehensive history had been taken from the patient, as she stated that dengue cases were present in her neighbourhood, with the latest case occurring one week before her symptoms commenced. The patient could not recall having any previous dengue infection. Rapid diagnostic tests (RDTs) for dengue non-structural protein 1 (NS1) antigen and Dengue Duo (WONDFO®) for immunoglobulins (IgM/IgG) were requested. The patient was positive for NS1 but negative for both IgM and IgG. The patient was then managed for suspected coinfection with dengue fever and COVID-19.On the second day in hospital, the patient received a positive result from the COVID-19 RT–PCR (FAM/ORF1AB 36.46). A repeat of the complete blood count showed a sudden drop in the platelet count, from the initial 156 × 109/L to 85 × 109/L. There was persistent leukopenia at 2.85 × 109/L, with a notable increase in the lymphocyte count (from 18% to 37%). The patient consented to receiving favipiravir, started at 1800 mg (9 tablets twice a day) as a loading dose then reduced to four tablets twice a day for 13 days. Later tests showed further decreases in platelet counts, falling to 37 × 109/L on the fourth day of hospitalization . A repeat test for dengue IgM/IgG on day 8 of illness was positive for IgG but negative for IgM. A repeat chest X-ray on day 9 of illness showed regression of previously noted densities in both lobes . On the succeeding days, increasing trends in the number of platelets and leukocytes were noted. A further dengue IgM/IgG test on day 10 of illness yielded the same result as the previous test. After 10 days in hospital, the patient was discharged with her symptoms improved.A chest computed tomography (CT) scan showed posterior-basal pneumonia with features atypical of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and minimal pleural effusion on the right. On the fifth day of hospitalization (day 7 of illness) maculopapular rashes appeared over the patient’s lower extremity, with some areas of erythematous petechial confluence and islands of normal skin  and viral culture are the ideal laboratory tests for the diagnosis of dengue. However, the only diagnostic tests available to the institution were the RDTs for dengue IgM/IgG and the NS1 antigen. The Handbook for clinical management of dengue notes that diagnosis of dengue infection is confirmed by the detection of the virus, the viral genome or NS1 antigen, or by seroconversion of IgM or IgG (i.e. from negative to positive). (In the natural course of dengue, IgM appears a few days following the onset of fever, followed by detectable IgG from day 5 onwards. The patient in this case study had a persistent negative IgM assay despite seroconversion of IgG, suggesting probable secondary dengue infection. Although the patient could not recall a previous dengue infection, she resides in a locality where dengue cases occur year-round; thus, it is possible that she had an undetected primary dengue infection. Primary dengue virus infections are often asymptomatic, and 90% of cases of dengue fever with symptoms occur following a second exposure. (In the setting of a positive COVID-19 RT–PCR result, persistence of a positive IgM or IgG result on follow-up studies supports coinfection with dengue and COVID-19. (Studies on coinfections with SARS-CoV-2 and arboviruses are lacking, which is not surprising given that SARS-CoV-2 is a new disease. An extensive literature search on dengue and COVID-19 in the Philippines suggested that no previous case of dengue and COVID-19 coinfection has been reported in the country. The medical challenge of such coinfection lies in the similarity of the clinical and laboratory features of the two infections; (Informed verbal and written consent was given by the patient.


We predicted and analyzed potential miRNA targets through online bioinformatic tools. To validate the interactions between circCDYL and its targets, we performed RNA fluorescence in situ hybridization, biotin-coupled miRNA capture assay, and biotin-coupled probe pull-down assay. Tight junction protein l (TJP1) was proved to be the target gene of the predicted miRNA by dual-luciferase reporter assay. The expression level of TJP1 in Wilms’ tumor cells was identified via Western blot. We showed that circCDYL was downregulated in WT tissue compared with adjacent non-tumor tissue. Upregulation of circCDYL could reduce cell proliferation, migration, and invasion. Mechanically, circCDYL, functioning as a miRNA sponge, decreased the expression level of miR-145-5p and TJP1 3′UTR was validated as the target of miR-145-5p, facilitating the circCDYL/miR-145-5p/TJP1 axis. In conclusion, our study suggested circCDYL as a novel biomarker and therapeutic target for WT treatment.Circular RNAs (circRNA) have been reported to exert evident functions in many human carcinomas. However, the possible mechanisms concerning the circRNA in various tumors are still elusive. In this research, we analyzed the expression profile and biological functions of circular RNA CDYL  in Wilms’ tumor. Here, miRNA and gene expression were examined by real-time PCR in Wilms’ tumor tissues and cell lines. The functions of circCDYL and its potential targets to influence cell proliferation, migration, and invasion in Wilms’ tumor cells were determined by biological functional experiments  Wilms’ tumor  accounts for about 6% of childhood tumors and for 95% of pediatric kidney tumors, ranking the fifth in childhood malignancy. Most WT develop before the age of 3 years . The recCircular RNA (circRNA) is a novel subtype of non-coding RNA with covalently closed loop containing the joint point of its 3′ end and 5′ end . Althougin vitro and in vivo.In this study, through deep sequencing in WT tissues, we identified has_circ_0008285, also called circCDYL, which was downregulated in WT tissues. CircCDYL, a circular RNA derived from the fourth exon of CDYL gene, is mainly located in cytoplasm and had no intronic sequence, IREs and ORFA . PreviouThe WT cell line SK-NEP-1 and G401 was obtained from American Type Culture Collection  and was cultured using McCoy’s 5A modified medium (30-2007) containing 15% FBS.–2, once a week, for 4 weeks; Actinomycin D 15 g kg–1 D–1, once daily, for the first 5 days) performed before surgery. No patients received radiotherapy or targeted therapy before the operation. Guangzhou Women and Children’s Medical Center Ethics Committee approved this study (grant no. 43600). All patients’ legal guardians provided written informed consent prior to enrollment in the study. Resected tumors were frozen in liquid nitrogen immediately and stored at −80°C condition.A total of 25 paired WT tissues and matched adjacent tissues were obtained from patients who were diagnosed as WT by histopathological examination in the Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, between April 2017 and May 2019. Samples obtained were classified in accordance with the WHO criteria for WT. All the patients received neoadjuvant chemotherapy with a VA plan (Vincristine 1.5 mg m–ΔΔCt method.The nuclear and cytoplasmic RNAs were isolated according to the manufacturer’s instructions . Both qRRNase R (4 U per 2 mg RNA) was used to treat RNA samples for 20 min at 37°C . RNA abundance was tested by RT- qPCR.CircCDYL-overexpressing adenovirus was designed by Hanbio . MiR-145-5p mimics/inhibitors and small-interfering (si) tight junction protein 1 (TJP1) were designed and synthesized by GenePharma . According to the manufacturer’s instructions, Lipofectamine 3000  was used during transfection.After cells were supplemented using Cell Counting Kit-8 (CCK-8)  for 120 min, wavelength of 450 nm was selected to detect the absorbency of cells.In colony formation assay, the procedures were utilized in accordance with the publication .Briefly, 100 μl of 50 μM EdU  was added into individual wells containing transfected cells, and they were incubated at 37°C for 2 h. Fluorescence microscopy was utilized to detect the proliferation.After infected cells grew to 100% confluence in six-well plates, the monolayer was scratched by a sterile pipette tip. Then, PBS was utilized to wash the wells for three times and serum-free medium was added into the wells. At 0 and 24 h, the width of the wound was photographed using DM2500 bright field microscope . The ImageJ software was utilized to quantify the cell migration capacity.5) were placed with serum-free McCoy’s 5A modified medium to the upper wells (8 μm membrane). Full culture medium (500 μl) was added into the bottom well. After incubation at 37°C for 24 h, cells in the upper surface were removed. Methanol was utilized to fix the cells stuck to the lower surface. Crystal violet (0.1%) was used to stain the cells. Then, cells were photographed using a microscope at 100 × magnification .The lower chambers were precoated with 100 μl of Matrigel  for 30 min before the addition of medium to the chambers. Transfected cells  and detected by flow cytometry. After that, the cell cycle was analyzed using a FACS Calibur flow cytometer .n = 8) were used. The mice were subcutaneously injected with 107 G401 cells infected with circCDYL overexpressing adenovirus or control. Tumor volumes were tested 10, 15, 20, 25, and 30 days after injection.The animal studies were approved by the Animal Management Committee of Guangzhou Medical University. G401 cells were transfected with the circCDYL adenovirus and GFP vector. Athymic BALB/c nude mice  and Ago2 antibody  were utilized to conduct the assay, and the procedures were performed as described before .GenePharma  designed and synthesized the specific biotinylated probes to bind circCDYL’s junction area, and an oligo probe was synthesized as control. The sequences were as follows: circCDYL: 5′-CAATCCTTTCAACCTTTCCCGTTAAC-3′-Biotin, miR-145-5p: 5′- AGGGATTCCTGGGAAAACTGGAC-3′-Biotin, and Oligo: 5′-TGTCTGCAATATCCAGGGTTTCCGATGGCACC-3′-Biotin. The procedures were performed as described before .GenePharma  designed and synthesized the DNA oligo probes for circCDYL (5′-CAATCCTTTCAACCTTTCCCGTTAAC-3′-Cy5) and for miR-145-5p (5′-AGGGATTCCTGGGAAAACTGGAC-3′-FAM). All procedures were performed as described before .TJP1 antibody  was used to measure the expression of TJP1 in WT tissues. The procedures were performed as described previously .The procedures were performed as described previously . The prit-test, and two-way ANOVA. SPSS and GraphPad Prism analyzed the data, and p value < 0.05 was set as significance threshold.The data were presented as mean ± standard deviation. The experiments were performed thrice independently. Statistical significances were determined using chi-square test, Student’s The circCDYL expression was significantly downregulated in five WT samples compared with adjacent non-tumor tissues from the results of circRNAs deep sequencing . RT-qPCRin vivo assay was carried out to explore the function of circCDYL in tumor formation. The result showed that downregulation of circCDYL reduced tumor formation in G401 cells . Biotin-coupled miR-145-5p analogously captured more circCDYL than biotin-coupled NC. This result indicated that miR-145-5p could attach itself to circCDYL . TJP1 was one of the potential targets. The possible binding sites between TJP1 and miR-145-5p are shown in To investigate the effects of TJP1 on WT cells, we transfected si-TJP1 or si-CTL into SK-NEP-1 and G401 cells and the protein level of TJP1 was subsequently detected by Western blot. The results revealed reduced TJP1 expression . Cells tTo investigate whether circCDYL exerts anti-oncogenic effects by regulating TJP1 expression level, we co-transfected SK-NEP-1 and G401 cells with circCDYL adenovirus and miR-145-5p mimics. In the co-transfected cells, increased TJP1 proteins induced by overexpressing circCDYL were partially reversed . MoreoveWT is a common urinary system carcinoma, and its worldwide morbidity and mortality remain considerable. In this research, we firstly reported that the circular RNA circCDYL was downregulated in WT and that the overexpression of circCDYL suppresses proliferation, migration, and invasion in WT cells.in vitro and in vivo, suggesting that circCDYL exerts an anti-oncogenic effect in WT. CircRNAs are identified to be tissue-specific or cell type-specific (In recent years, accumulating studies have substantiated that abnormal expression of circRNAs is connected with the generation and progression of various diseases, and can be used as therapeutic targets and biomarkers . It was specific , which mPrevious studies demonstrated that circRNAs can exert as a ceRNA of binding to the specific miRNAs, due to their specific miRNA response elements, and to ulteriorly regulate the target genes . MiRNA iin vitro (miR-145-5p was reported to have antioncogenic properties in several tumors. Previously, the levels of miR-145-5p were reported to be lower in colorectal cancer and decreased miR-145-5p expression promoted the migration, invasion, and epithelial–mesenchymal transition . Additioin vitro . It was in vitro . Our resin vitro . It was in vitro . Targetiin vitro . In our In conclusion, our research demonstrated that circCDYL can sponge miR-145-5p to upregulate TJP1 expression level, enabling it to restrain cell proliferation, migration, and invasion. These findings indicate that circCDYL/miR-145-5p/TJP1 may be a novel therapeutic target for WT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Guangzhou Women and Children’s Medical Center Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The animal study was reviewed and approved by the Animal Management Committee of Guangzhou Medical University.GL and WF designed the study. RZ, WJ, and XG collected the clinical information, performed the experiments, and wrote the manuscript. FD, KF, TZ, and ZL helped in conceiving and/or analyzing the experiments and provided reagents. All authors read and approved the manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work were appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In recent years, curcumin has been a major research endeavor in food and biopharmaceutical industries owing to its miscellaneous health benefits. There is an increasing amount of research ongoing in the development of an ideal curcumin delivery system to resolve its limitations and further enhance its solubility, bioavailability and bioactivity. The emergence of food-graded materials and natural polymers has elicited new research interests into enhanced pharmaceutical delivery due to their unique properties as delivery carriers. The current study is to develop a natural and food-graded drug carrier with food-derived MCT oil and a seaweed-extracted polymer called k-carrageenan for oral delivery of curcumin with improved solubility, high gastric resistance, and high encapsulation of curcumin. The application of k-carrageenan as a structuring agent that gelatinizes o/w emulsion is rarely reported and there is so far no MCT-KC system established for the delivery of hydrophobic/lipophilic molecules. This article reports the synthesis and a series of in vitro bio-physicochemical studies to examine the performance of CUR-MCT-KC as an oral delivery system. The solubility of CUR was increased significantly using MCT with a good encapsulation efficiency of 73.98 ± 1.57% and a loading capacity of 1.32 ± 0.03 mg CUR/mL MCT. CUR was successfully loaded in MCT-KC, which was confirmed using FTIR and SEM with good storage and thermal stability. Dissolution study indicated that the solubility of CUR was enhanced two-fold using heated MCT oil as compared to naked or unformulated CUR. In vitro release study revealed that encapsulated CUR was protected from premature burst under simulated gastric environment and released drastically in simulated intestinal condition. The CUR release was active at intestinal pH with the cumulative release of >90% CUR after 5 h incubation, which is the desired outcome for CUR absorption under human intestinal conditions. A similar release profile was also obtained when CUR was replaced with beta-carotene molecules. Hence, the reported findings demonstrate the potencies of MCT-KC as a promising delivery carrier for hydrophobic candidates such as CUR. Curcuma longa and is responsible for the yellow pigmentation of turmeric. Given its antimicrobial, antioxidant, antitumor, anti-inflammatory, and immunostimulatory properties, it has become one of the most widely studied food-derived compound [Curcumin (CUR) is a biologically active, non-polar, and naturally occurring polyphenolic compound from turmeric that possesses a variety of health benefits . It is tcompound . BecauseDespite its health benefits, its usage is limited by its poor water solubility, stability, and bioavailability ,4. CUR uOne of the most suitable modes of delivery for CUR is through emulsions. Both CUR nano- and micro-emulsions have been extensively applied in drug delivery, the cosmetic sector, and food industry, for effective delivery of hydrophobic molecules . HoweverMedium-chain triglycerides (MCT) and KC employed in this study are generally recognized as safe (GRAS), non-toxic, edible, biodegradable, biocompatible, and biologically safe. These are interesting features for biopharmaceutical and food industries, owing to the contemporary direction of green consumerism. CUR in an edible medium such as MCT oil and KC, can be directly used in food or pharmaceutical ingredients, without the requirement of eliminating the extraction medium. MCT are triglycerides with two or three fatty acids and a short chain length of 6–12 carbon. It can be extracted from food sources such as coconut oil and palm kernel oil . MoreoveRhodophyceae [KC, on the other hand, is a linear, anionic, hydrophilic, and sulphated polygalactan naturally derived from red seaweeds of the class ophyceae . It is mophyceae . KC compophyceae ,15,16,17ophyceae  and it hophyceae ,20. Furtophyceae .In the present study, a modified O/W single emulsion technique is reported to prepare the CUR-MCT-KC with better properties by using MCT as a suitable oil phase for CUR encapsulation and KC as the aqueous phase that gelatinizes CUR-MCT emulsion into gel beads. Emulsion is a kinetically stable, non-equilibrated, and colloidal system comprising two or more immiscible liquids. There are various studies that have reported the use of either MCT or KC natural compounds for the encapsulation, delivery, and stabilization of CUR ,24,25,26This research work, the first of its kind, was able to fabricate CUR-MCT-KC gel beads and evaluate the efficiency of MCT oil and KC polysaccharides for CUR emulsification and high gastric resistance. The aim of this study was to design and develop a natural, food-graded oral delivery system for the delivery of CUR and examine the synergetic effects of MCT-KC for better encapsulation, solubility, and release of CUR. The in vitro experiments were carried out on the hypothesis that a MCT-KC nature-derived formulation can stabilize, encapsulate, and deliver CUR effectively via the gastrointestinal tract (GIT) when administrated orally. The differences between CUR-KC, CUR-MCT, and CUR-MCT-KC were also examined and compared to demonstrate the potential of CUR-MCT-KC formulation. This work can therefore provide useful in vitro information for the development of an oral delivery system of other nutraceuticals, with poor solubility to further enhance their loading capacity and practical applications.Curcuma longa (Turmeric), powder), KC (sulfated plant polysaccharide), phosphate-buffered saline (PBS) , potassium chloride (KCl) , acetonitrile (ACN) , dichloromethane (DCM), bile salts , Tween 20 (polysorbate 20), and Span 20 (sorbitan monolaurate) were purchased from Sigma Aldrich, Singapore. MCT oil (Neobee 1053) was purchased from Stepan company, Northfield, IL, USA. Deionized (DI) water was used in all the experiments.Curcumin  via homogenization at 80 °C with 13,000 rpm for 5 min.Initially, 5 mg CUR was dissolved in 1 mL MCT oil and Span 20 (10%) by stirring the mixture at 60 °C with gentle shaking at 150 rpm for 5 min or until an orange solution was formed; 60 °C was adopted because it is a temperature that can be replicated in industrial conditions. The temperature was increased to 120 °C if the CUR was not dissolved completely; 2% Finally, the CUR-MCT-KC bead production was conducted via a crosslinking process using KCl as crosslinkers. The above-prepared CUR-MCT-KC solution was dropped through a 1 mL syringe with a needle into a beaker containing a 5% KCl aqueous phase. Beads were formed and collected in the aqueous phase, resulting in spherical CUR-MCT-KC beads, as displayed in w/v CUR-MCT solution) before mixing with 0.4% w/v KC solution. On the other hand, beta-carotene-MCT-KC (BC-MCT-KC) beads were also fabricated using the same techniques and procedures by replacing CUR with BC molecules.Similar procedures were carried out for the fabrication of CUR-MCT-KC beads containing a higher concentration of MCT (CUR-MCT-KC_2) using 5 mL MCT to dissolve 5 mg CUR  was used to visualize the size and surface morphology of the CUR-MCT-KC bead. SEM is a technology that uses the focused electron beams and different signals to obtain the sample image. The samples were mounted onto carbon tape prior to the sputter coating with gold for 45 s. Each sample was then imaged at different magnification of 35×, 50×, 150×, 550×, and 1000× with a 5 kV voltage and 20 mm working distance.® M200 . To ensure the measurement accuracy, 300 µL volume was standardized for every analysis made. Moreover, the wavelength used for BC measurements was 448 nm.The presence of CUR can be identified by the maximum absorption peak that can be determined using UV-visible spectrophotometry at the wavelength of 425 nm. This peak is mainly due to the π–π type excitation of the CUR aromatic system . The proThe thermal stability and behavior of CUR-MCT-KC beads was investigated using TGA  to evaluate the change in mass at a constant heating rate in an inert environment. This is essential to determine the thermal stability and indicate the potential molecular rearrangement within the matrices of the CUR-MCT-KC complexation in terms of exotherm. The TGA instrument was purged with nitrogen at 20 °C/min and equilibrated at 25 °C prior to the analysis. Approximately 13 mg CUR-MCT-KC was placed in an aluminum pan and analyzed from 30 to 600 °C with 10 °C per min of constant heating rate and 25 mL/min of nitrogen gas. STARe software was applied for the result analysis.−1 at a 4 cm−1 resolution. Sixteen scans for each sample were conducted to lower the sound-to-noise ratio.In order to indicate the formation of CUR-MCT-KC and the presence of functional groups in CUR-MCT-KC, CUR, MCT oil, and CUR-MCT, prepared samples were characterized using the Fourier Transform Infrared Spectroscopy (FTIR) . Potassium bromide (KBr) was utilized as the background pellet and each sample  was ground with the KBr in the ratio of 1:4 to form pellet using the hydraulic press. Spectra were measured in the range of 400–4000 cmThe quantity of CUR encapsulated within MCT-KC was evaluated from the difference between the initial amount of CUR added in the formulation and the amount of free CUR measured in the medium upon the breakdown of MCT-KC. Next, 10 mg CUR-MCT-KC bead was added into 1 mL distilled water and heated at 60 °C for 5 min or until a clear and homogenous solution was obtained and 1 mL ACN was added to the mixture prior to centrifugation at 13,000 rpm for 3 min; 300 µL supernatant was then collected for UV-spectrophotometry analysis at 425 nm. All absorbance readings were measured in triplicate and averaged. The CUR concentration was determined using the standard curve of CUR in ACN. The encapsulation efficiency (EE) was calculated from the following equation:In 1 mL MCT oils, an excess amount of CUR, 10 mg was added and heated to 60 °C under stirring to ensure complete dissolution of CUR in MCT. The temperature was increased to ~120 °C if the CUR was not dissolved completely. After cooling to 25 °C, the mixture was centrifuged at 13,000 rpm for 5 min to collect the supernatant (CUR-saturated MCT oil) prior to the UV-spectrophotometry analysis at 425 nm. The following equation was used to calculate the loading capacity:The effect of heat on the CUR solubility in MCT oil was studied over a temperature range of 37–100 °C using a water bath. 1 mg/mL CUR was prepared using MCT and heated from 37 to 100 °C. At specific intervals , samples were collected to be examined spectrophotometrically at 425 nm, as stated in A dissolution study is essential to understand the solubility of naked/unformulated CUR as compared to a CUR-MCT mixture in PBS buffer that mimics the human physiological environment at pH 7.4, temperature 37 °C, and 150 rpm in a shaking incubator. Two different samples were prepared: (1) 4.15 µg/mL CUR solution prepared using PBS; the saturated CUR concentration reported in PBS; (2) 1.32 ± 0.03 mg/mg CUR-MCT solution prepared using MCT; the saturated CUR concentration in MCT determined from our study. Samples were mixed with 5 mL PBS (+0.02% Tween 20) and incubated at 37 °C, 150 rpm for 1 h. At every time interval , samples were collected for centrifugation and UV-spectrophotometer measurements at 425 nm. The CUR concentration was determined using the standard curve of CUR in PBS. The percentage of CUR dissolved was calculated from the following equation:The effect of pH on encapsulation efficiency of CUR-MCT-KC and CUR-KC was evaluated and compared via the in vitro release study at extreme acidic and alkali pHs: pH 1.2 and pH 7.4. 2 mg of the CUR-MCT-KC bead was incubated in 1 mL PBS (+0.02% Tween 20) at pH 1.2, 150 rpm, and temperature 37 °C for 2 h in a shaking incubator. At every time interval , a supernatant was collected to be measured spectrophotometrically at 425 nm to identify the amount of released CUR. Fresh PBS was added to replenish the extracted sample at every time interval; the pH of PBS was adjusted using 0.1 M sodium chloride (NaCl) or 0.1 M potassium hydroxide (NaOH) to the respective pH level. The above procedures were repeated for CUR-MCT-KC bead under the alkali condition at pH 7.4, and a release study of CUR-KC bead at both pH 1.2 and pH 7.4. The percentage of CUR released was calculated from the following equation:The release mechanism of encapsulated CUR under simulated GI conditions was examined by mimicking the physiological environment in the upper tract  of human GIT. The CUR-MCT-KC gel beads (2 mg) were incubated in 1 mL simulated gastric fluid (SGF) +0.02% Tween 20 at pH 1.2, temperature 37 °C, 150 rpm in a shaking incubator for 2 h, which represents the average transition time of GI. The entire sample after 2 h of gastric digestion was then transferred to the simulated intestinal fluid (SIF) +0.3% bile salts for a subsequent incubation of 3 h. At specific time intervals , 1 mL supernatant was collected and mixed with 1 mL DCM prior to centrifugation at 13,000 rpm for 3 min. This is to separate the released CUR from the loaded beads. Free CUR is very soluble in organic solvent–DCM. The CUR-DCM supernatant was collected and vacuum-dried. The released CUR was re-dissolved in 1 mL ACN to assay spectrophotometrically at 425 nm. All absorbance readings were measured in triplicate and averaged. Fresh SGF or SIF was added to replenish the extracted sample at every time interval. The concentration of released CUR was then determined using the standard curve of CUR in ACN. The percentage of CUR released was calculated from the following equation:The above procedures were replicated for in vitro release study of CUR-MCT-KC, CUR-MCT emulsion in simulated GI conditions, and BC-MCT-KC beads in different conditions: (1) SGF for 5 h; (2) SIF for 5 h; (3) SGF for the first 2 h and SGF for the subsequent 3 h. Chloroform was used instead of DCM in the extraction of BC due to its solubility in different organic solvents. Moreover, the wavelength used for BC measurement was 448 nm.Each block of experiment was conducted in triplicate (minimum), and the average measured values were reported as the final analytical data. Experimental data are recorded as average ± standard deviation and/or standard error.Based on the literature, the use of natural oils  and natural polymers such as KC, as a delivery carrier for hydrophobic CUR molecules ,34,35,36SEM was utilized to examine the size and surface morphology of the prepared CUR-MCT-KC formulation at different magnifications of 35×, 50×, 150×, 550×, and 1000× in CUR-MCT-KC_2 that was comprised of a higher amount of MCT was shown to be less spherical in shape with smoother and less porous surface features, as displayed in The thermal behavior and stability of CUR-MCT-KC was studied using the TGA characterization. FTIR spectrum indices were used to differentiate between CUR, MCT, KC, and indicates the formation of CUR-MCT-KC formulation. The FTIR spectrum and vibrational characteristics of various functional groups presented in each component  were identified as displayed in −1, 1603 cm−1, 1627 cm−1, 1023 cm−1, and 724 cm−1 attributed to the phenolic (OH), carbonyl C=O and alkenes C=C, benzene ring, C-O-C stretching vibrations, and CH2 stretching vibrations of CUR, respectively. On the other hand, the chemical interactions and the formation of CUR-MCT-KC were confirmed, as shown in −1 peak with slight spectral changes due to interactions of CUR and MCT. On top of that, other functional groups associated with 1635 cm−1, 1605 cm−1, and 1038 cm−1 peaks showed the encapsulation of CUR; 2923 cm−1, 1743 cm−1, and 1466 cm−1 identified the incorporation of MCT, and 922 cm−1, 845 cm−1, and 701 cm−1 revealed the presence of KC. Hence, the presence of CUR, MCT, and KC functional peaks in the FTIR analysis indicates the conjugated composite materials of CUR-MCT-KC beads.Regarding the solubility and encapsulation of CUR in MCT to form CUR-MCT, the FTIR spectra indicates most of the active functional groups of CUR have been introduced in MCT. This includes 3504 cmEE is the percentage of CUR being entrapped successfully into the MCT-KC formulation. The CUR-MCT-KC_2 bead exhibited a significantly higher EE (73.98 ± 1.57%) than CUR-MCT-KC bead (~24.04 ± 2.17%). EE is therefore relatively dependent on the amount of MCT oil used. This also indicates that the use of MCT can improve the solubility of CUR to a greater extent. Their storage stability was also examined with CUR-MCT-KC_2 beads showing a good stability of up to at least 15 days at room temperature (EE of 69 ± 0.034%), and 30 days of storage at −20 °C (EE of 71 ± 0.018%). Hence, CUR-MCT-KC_2 beads were selected for further investigations in terms of solubility, dissolution, and drug release studies.The surface charge and structural diversity of the encapsulation materials used can notably affect their efficiency in encapsulating and retaining CUR. Initially, CUR interacted with the fatty acid chains of MCT that aided in its solubilization. Ma, Zeng  suggesteBesides this, the emulsifier plays an important role in the formation of a stable emulsion. Span 20 with an intermediate hydrophilic-lipophilic balance value of 7−9 was used in this study. It was determined to improve the CUR solubility and effectively prevent CUR crystallization, even after being stored at room temperature for months . The preThe EE 73.98 ± 1.57%) of CUR-MCT-KC_2 bead is highly comparable and performs better than other reported CUR-loaded formulations including CUR-MCT nanoemulsion with 71.5% EE , CUR-KC 8 ± 1.57%The solubility of CUR in MCT oil was examined in this study to understand the loading capacity of CUR per ml of MCT used. The result reveals that the solubility of CUR in MCT was 1.32 ± 0.03 mg/mL, which is more than 100 times higher than the solubility of unformulated CUR in PBS (4.15 µg/mL) . This caCUR molecules were readily dispersed in MCT oil with increased heat, as seen in CUR is not soluble in the water phase and its solubility is extremely low, even with the application of emulsifiers. Dissolution is defined as the rate of solute dissolving in a solution where it is a kinetic process and is measured by its rate. A simple in vitro experiment was carried out to determine the amount of CUR dissolved in PBS after an incubation period of 1 h, with/without the presence of MCT. PBS was used in the study to mimic the human physiological environment. Unlike the unformulated or naked CUR, MCT-solubilized CUR was shown with a greater in vitro dissolution rate in PBS. The data, as seen in The unformulated CUR in PBS started to degrade gradually and only around 22% was detected after 1 h of incubation as compared to its initial amount added. The poor solubility of unformulated CUR (~33%) is in keeping with other studies, which revealed the high hydrophobicity and poor solubility of unformulated CUR ,48. ThisThe digestion of delivery carrier in the GIT is a complex mechanism and its impact on the release of encapsulated bioactive compounds plays an important role in the uptake, distribution, and bioavailability of the encapsulated compound. Both MCT and KC were evaluated for their entrapment efficiency for CUR via the in vitro release study under human physiological conditions. This study is important to demonstrate the synergetic effects of both MCT and KC as an oral delivery carrier. The conditions of an incubating medium could crucially affect the drug release profile. Hence, both the gastric and intestinal digest were investigated in vitro for the cumulative release of CUR from MCT-KC beads, as seen in The optimal hydrolysis of KC is largely dependent on the pH of the reaction mixture. As displayed in + ions [+), calcium (Ca+), or K+ ions strongly enhances the stability of the ordered helices and aggregations, which further promotes gelation, resulting in a slower depolymerization rate of KC in acidic condition. This describes the effectiveness of fabricating MCT-KC via a KCl crosslinking process in this study to obtain a more solidified carrier for hydrophobic molecules with better protective outcomes. Based on In this study, KCl was demonstrated as an effective crosslinker whereby KC became more resistant to acid hydrolysis in the stomach upon the crosslinking with K+ ions . The preIn the initial phase, the release (%) of CUR from CUR-MCT-KC was gradual and there was no initial burst observed at the first hour of incubation in SGF at 37 °C for 2 h, indicating that CUR-MCT-KC was relatively resistant to pepsin digestion. This ensures that the encapsulated CUR is delivered and released in the targeted intestinal region. When incubated in SIF for the following 3 h, CUR-MCT-KC was capable of releasing the maximum amount (>90%) of entrapped CUR between the third and the fourth hour of incubation in SIF, as seen in The release pattern of CUR-MCT-KC has specified its suitability for oral administration of drugs. Oral delivery is one of the most preferred routes, due to its high patient compliance. As shown in Lactobacillus plantarum and Lactobacillus rhamnosus, as well as other poorly soluble drugs for enhanced GIT delivery [The release profile of the CUR-MCT-KC bead was compared to that of the CUR-MCT emulsion, as illustrated in delivery ,60,61,62In this study, the pH used to mimic the gastrointestinal condition was extreme, at around pH 1.2. This was to investigate the protective effect of KC under the most unfavorable and rapid acidification of gastric digestion. In fact, under the in vivo condition or the human physiological environment, the gastric of GIT is acidified gradually, whilst gastric emptying is initiated as soon as food ingestion takes place. The gastric pH of a fasted- and fed-stated is ~pH 1.3 and pH 5, respectively in healthy subjects due to the buffering effects of ingested meals ,64. HencPancreatic enzymes, also known as pancreatin, are commercial mixtures of lipase, protease, and amylase enzymes . When CUThe swelling ability of CUR-MCT-KC beads were larger in pH 6.8 compared to pH 1.2, due to the changes in the ionic structure of KC when exposed to a different medium. It is believed that at pH 1.2, the hydrogen bonds between CUR and KC molecules are much stronger due to the existence of a carboxylic group (COOH) of polymers and OH groups which limit swelling whilst electrostatic repulsions are intensified between ionized groups  at pH 6.8 of intestinal fluid, leading to greater swelling effects. Consequently, media diffusion into the beads is higher and resulting to a higher release of encapsulated CUR in SIF .In SIF, the presented bile salts play a role in changing the interface, which facilitates the lipase-based degradation and thus enhances CUR release. It was reported that MCT is highly favorable over LCT, because MCT can be directly absorbed via the portal vein to the liver without the formation of chylomicrons . This maIt is proposed that released CUR from hydrolyzed MCT conjugates can then diffuse through enterocytes more rapidly to interact with other lipid moieties to achieve a higher lymphatic drug transport. This is more effective than CUR-LCT conjugates. Theoretically, there are two possible mechanisms which occur to release the CUR molecules upon MCT lipolysis. MCT lipolysis releases a higher amount of CUR within a short time. Released CUR molecules attach and diffuse passively across enterocytes with a greater uptake efficiency in a shorter time due to its small size. Hydrophobic CUR molecules can then be solubilized with other fatty acids or monoglycerides into chylomicrons to be diffused into the lipid absorption pathways towards systemic blood circulation via the intestinal lymphatic transport, which bypassed the first-pass metabolism in the liver . This reThere was no initial burst at the first hour for CUR-MCT-KC formulation. Although the burst release of CUR-MCT was higher (approximately 35%), MCT oil is still capable of providing some shielding effect to the encapsulated CUR as compared to naked CUR molecules, which are susceptible to gastric digestion. A higher release was observed at the second hour from the CUR-MCT emulsion (>50% cumulative release) as compared to CUR-MCT-KC beads (<30% cumulative release), indicating the shielding effect of KC under an extreme acidic environment. Data are expressed as the mean ± standard error of three independent experiments.To achieve a comparable encapsulation and desired GIT drug release pattern, MCT-KC delivery system was also used to encapsulate another drug model, BC to examine the potency of MCT-KC formulation in delivering different hydrophobic molecules. A similar release trend was observed in The ultimate use of natural polymers and food-grade composites has become an attractive approach in current pharmaceutical delivery applications, attributed to their biological origin, biodegradability, and non-toxicity. In this study, CUR was formulated into MCT-KC to improve its solubility, encapsulation, and acid resistance. FTIR spectra and SEM images have confirmed the formation of the CUR-MCT-KC formulation. MCT-KC has also demonstrated its capability to efficiently encapsulate and release CUR under the simulated upper tract GI conditions. KC was shown with improved acid resistance at extreme pH 1.2 and simulated gastric condition, indicating its potency in offering an additional shielding effect to the encapsulated CUR. The solubility of CUR was improved using MCT two-fold with an encapsulation efficiency of 73.98 ± 1.57% and a loading capacity of 1.32 ± 0.03 mg CUR/mL MCT, suggesting the suitability of MCT as a carrier for hydrophobic compounds. The release profile of MCT-KC has revealed the delivery system with no premature burst of CUR and suppressed release under extreme simulated gastric condition (<35% release), followed by a drastic release of CUR in the mimicked intestinal condition (~100% release after 3 h incubation), which is favorable for further CUR absorption into the systemic circulation. CUR-MCT-KC beads can be stabled for at least one month at −20 °C storage conditions. On the whole, findings from this work has demonstrated CUR loaded MCT-encapsulated KC formulation which could be potential materials for the development of natural, food-graded and polymer-derived delivery vehicles with enhanced characteristics for pharmaceutical applications. Further in vivo research will be carried out to explore more valuable functions and the potential of CUR-MCT-KC as an oral delivery system.